The invention refers to methods of examining biological, biochemical, and chemical characteristics of media, mainly of biological origin, or media that are in contact with biological objects whose living is influenced by the media characteristics.
In a known analogue [B. Liedberg, C. Nylander, and I. Lundstrom, Surface plasmon resonance for gas detection and biosensing, Sensors and Actuators, 4 (1983) 299-304] of the proposed method, a solution that contains an antigen is brought into contact with a thin layer of antibodies immobilised on a silver film adjacent to a glass prism. The film is exposed to laser radiation incident through the prism. Surface plasmon polaritons (SPP) are excited at the antibody layer-silver interface. One observes a resonant minimum in the reflected radiation intensity dependence on the angle of the radiation incidence on the film. The minimum is due to the pumping of radiation power into that of SPP. The interaction of the antigen and the antibody is recorded as a shift of the resonant contour of the dependence. The drawbacks of both the method and the apparatus of the analogue [B. Liedberg, C. Nylander, and I. Lundstrom, Surface plasmon resonance for gas detection and biosensing, Sensors and Actuators, 4 (1983) 299-304]. are associated with that it necessitates mechanical rotation units to scan and adjust the incidence angle, as well as to compensate for a displacement of the irradiation spot and to follow a rotation of the reflected beam. This makes the method and the apparatus cumbersome and unpractical, results in insufficient reliability, low accuracy of measurements, and weak sensitivity of the method.
In another analogue [WO 89/07252, G01N 21/17, 1989], radiation is fed into an optical waveguide with the output face bevelled at an angle that ensures the excitation of SPP at the interface of a sensitive layer and a metal film deposited on the face. The layer is capable of reacting with the medium component under test and changing by this means the conditions of the resonant SPP excitation. An information signal is extracted from the analysis of the radiation reflected back into the waveguide. The drawbacks of the analogue [WO 89/07252, G01N 21/17, 1989] are complexity of the method and the apparatus, connected with the techniques and means to analyse the output optical signal, and the need for selection of radiation modes and frequencies. These restrict the areas of application, lowers the accuracy of measurements and the sensitivity of the method.
The closest to the proposed invention is the analogue method of examining biological, biochemical, and chemical properties of media [EP 0 305 109 B1, G01N 21/55, 1993]. It comprises:
introducing a volume or a constituent of a medium under test into the region where it interacts with a sensitive material;
acting by electromagnetic radiation through a block transparent to the radiation on a metal layer located on a boundary surface of the block, said sensitive material being placed over the metal layer directly or on an intermediate material;
exciting surface plasmon polaritons by means of said acting;
reflecting partially said radiation from the surface of said metal layer, resulting in the formation of a beam of reflected electromagnetic radiation;
producing with said beam such a spatial distribution of electromagnetic field intensity that the distribution comprises features whose positions depend on the interaction of the medium under test with said sensitive material;
recording parameters of said distribution, from comparison of which with predetermined reference relationships the examined characteristics are judged.
The basis of the method is that in a spatial distribution of electromagnetic intensity that is formed using the reflected beam over an extended photodetector array there is a feature associated with the excitation of SPP, namely, a resonant intensity minimum. In a one-dimensional distribution, it is revealed as a dark band on the illuminated background area. The method of the analogue allows to record the spatial intensity distribution with the resonant contour of the reflectance minimum as a whole at every instant of time and obtain information on characteristics under study by the analysis of the position and the shape of the resonant contour. In so doing the method avoids mechanical rotations and displacements. Besides, the output signal is insensitive to radiation intensity drifts. The mentioned features are among important advantages of the analogue.
The main drawback of the analogue is low sensitivity of the output signal to variations in optical parameters of the sensitive material layer. This results in low resolution of the method. As reported in literature, such schemes enable one to achieve resolution no better than 3xc3x9710xe2x88x926 in terms of effective refraction index, and 10xe2x88x928 in terms of albumin aHSA concentration detected by immunological binding HSA-aHSA directly on a gold surface. However, there is a number of problems in which lowering of a detection limit of biologically active components is of crucial importance. The example is hepatitis virus detection since even a single virus can cause infection. But, fundamental limitation on the prototype""s resolution limit is imposed by the physical principle used, namely, sensitivity of the spatial position and/or the level of the reflected intensity minimum to variations in optical parameters of the sensitive material layer.
Besides, the detection of shifts of the position or the level of a resonant minimum involves the necessity to record all the resonant contour or the most part of it. The reason is that it is difficult to describe analytically the shape of the contour and actually impossible to find the position and the level of the minimum from few points of the contour. Thus, the detectability threshold of small shifts of the resonant contour is the lower, the greater is the spatial scale of the produced intensity distribution and the less is the size of each discrete element of the extended photodetector array. Since the angular width of the resonant contour is a fixed value defined by the physical mechanism of SPP excitation, lowering of the detectability threshold can be achieved only at the expense of increasing the spatial scale (and, consequently, the size of the photodetector array and the overall apparatus) or decreasing the size of each element of the array. Both approaches lead to a raise in the cost of the method and the apparatus, as well as to a fall in signal-to-noise ratio, and appear to be hardly acceptable.
Thus, the required technical result that eliminates the drawbacks of the known methods consists in rising the sensitivity and lowering the resolution threshold of the method, or, more concretely, in the following:
a) taking advantage of a superior physical principle to yield a parameter to be measured, which pertains to a spatial electromagnetic intensity distribution, so that the principle would ensure a higher sensitivity of this parameter to the characteristics of media under examination;
b) taking advantage of a more flexible technique to record small variations of said parameter, which would allow to lower the detectability threshold.
The known methods described above have been embodied in the apparatus for examining biological, biochemical, chemical characteristics of media. Their drawbacks are mentioned above as well as the required technical results eliminating them. The closest to the proposed apparatus is an analogue apparatus [EP 0 305 109 B1, G01N 21/55, 1993]. It comprises:
a source of electromagnetic radiation directed through a block transparent to the radiation on to a metal layer located on a boundary surface of said block so that there takes place a configuration for excitation of surface plasmon polaritons and partial reflection of said radiation from the surface of said metal layer, with formation of a reflected radiation beam;
a sensitive material placed over said metal layer directly or on an intermediate material;
a unit for introducing a volume or a constituent of a media under test into the region where it interacts with said sensitive material, the region being situated so that said interaction influences the properties of said surface plasmon polaritons and said reflected radiation beam;
a means for producing, with the use of said reflected radiation beam, a spatial electromagnetic field intensity distribution that contains features whose positions depend on said interaction;
a block for recording parameters of said distribution to obtain on its base an output information signal.
The known apparatus operates as follows. Radiation from a source is incident through a transparent block (which contains, for example, a glass prism and a glass slide in immersion contact) on a metal layer located on its boundary surface at the angle that ensures SPP excitation according to the frustrated total internal reflection configuration. On the metal surface there is a layer of a sensitive material. A volume or a constituent of the medium under analysis is introduced into contact with the sensitive material. All the arrangement is chosen so that the interaction of the medium under analysis with the sensitive material affects the properties of SPP and a beam that is formed due to a partial reflection of the incident radiation. Particularly, dependent of medium properties is the complex wavevector of SPP, which determines the position and the shape of a resonant contour with a minimum in the angular dependence of the reflected radiation intensity. To record the resonant contour, in the prototype apparatus one employs a means for producing a spatial intensity distribution with the use of the reflected radiation beam. The means comprises components to specify the spatial width of the incident radiation beam and to focus the beam on the metal layer so that a range of incidence angles is provided, that embraces the resonant contour or its part. After a partial reflection, a divergent beam is formed, which comes to a photodetector that is capable of receiving a range of angles necessary for obtaining information on medium characteristics under test from the features of the resonant contour of the reflected radiation intensity. The example is an extended photodetector array that consists of a large number of discrete photosensitive areas (pixels), where the position of the resonant minimum of the reflected beam intensity is expressed as the number of such an area (pixel). The information yield is obtained from the analysis of the position or/and the level of the resonant intensity minimum.
The known apparatus has the drawbacks described in detail above, in the discussion on the known method. Briefly, they can be summarised as low sensitivity and insufficient resolution. Besides, as the angular width of the resonant contour is fixed, to lower the resolution threshold of the shifts of the spatial intensity distribution one has to enlarge the size of the photodetector array and the overall apparatus, as well as the number of photosensitive pixels with decreasing their size. This leads to a dramatic raise in the cost of the apparatus and a fall in a signal-to-noise ratio.
To achieve the technical result stated above, there is proposed a method of examining biological, biochemical, chemical characteristics of media, including characteristics of media interactions with surfaces and superficial layers, which comprises:
introducing a volume or a constituent of a medium under test into the region where it interacts with a sensitive material;
acting by electromagnetic radiation through a block transparent to the radiation on a metal layer located on a boundary surface of the block, said sensitive material being placed over the metal layer directly or on an intermediate material;
exciting surface plasmon polaritons by means of said acting;
reflecting partially said radiation from the surface of said metal layer, resulting in the formation of a beam of reflected electromagnetic radiation;
producing with said beam such a spatial distribution of electromagnetic field intensity that the distribution comprises features whose positions depend on the interaction of the medium under test with said sensitive material;
recording parameters of said distribution, from comparison of which with predetermined reference relationships the examined characteristics are judged;
in a manner like the analogue.
The proposed method differs in that said distribution is produced using interference of said beam and, at least, one more beam of electromagnetic radiation, which differs from the former beam in position and/or direction in space anywhere over its preceding propagation path.
In addition, said distribution may be produced with two beams reflected from the surface of said metal layer so that the properties of only one of the beams depend on the interaction of the medium under test with said sensitive material.
In addition, both said beams may be formed under conditions of surface plasmon polariton excitation.
In addition, one may use non-monochromatic radiation with a discrete and/or continuous set of frequencies inherent in said radiation, and parameters of said distribution may be recorded at a number, or within a band, of frequencies that belong to said set.
In addition, said beam contains radiation components with mutually orthogonal polarisation directions and said distribution is produced using interference of the beams comprising said components.
In addition, said electromagnetic radiation acting on said metal layer is shaped as a divergent or convergent beam.
The method described above has been embodied in a proposed apparatus for examining biological, biochemical, chemical characteristics of media, including characteristics of media interactions with surfaces and superficial layers. It eliminates the mentioned drawbacks of the analogue apparatus, and comprises:
a source of electromagnetic radiation directed through a block transparent to the radiation on to a metal layer located on a boundary surface of said block so that there takes place a configuration for excitation of surface plasmon polaritons and partial reflection of said radiation from the surface of said metal layer, with formation of a reflected radiation beam;
a sensitive material placed over said metal layer directly or on an intermediate material;
a unit for introducing a volume or a constituent of a media under test into the region where it interacts with said sensitive material, the region being situated so that said interaction influences the properties of said surface plasmon polaritons and said reflected radiation beam;
a means for producing, with the use of said reflected radiation beam, a spatial electromagnetic field intensity distribution that contains features whose positions depend on said interaction;
a block for recording parameters of said distribution to obtain on its base an output information signal;
in a manner like the analogue.
The proposed apparatus differs from the analogue in that said means for producing a spatial electromagnetic field intensity distribution comprises a facility for separating radiation into; at least, two beams, a first of them comprising radiation that participates in the formation of said reflected radiation beam, and a second one differing from the first beam in position and/or direction in space, as well as a facility for bringing radiation from said first beam and from said second beam to an area where interference of radiation from these beams occurs, the position of said block for recording parameters of said distribution being appropriate to the position of said area of interference.
Besides, the apparatus is made so that it is allowed to vary the angle that defines the direction of radiation from said source of electromagnetic radiation with respect to said metal layer. This is necessary for the adjustment at an operation point of the incidence angle or recording the resonant contour of the angular dependence of reflected beam intensity.
Besides, said source of electromagnetic radiation may allow to specify a discrete or continuous set of frequencies of the outgoing radiation, and said block for recording parameters of said distribution may allow to perform said recording at a number, or within a band, of frequencies that belong to said set. This is appropriate for recording resonant features of reflected beam intensity against frequency rather than incidence angle. In particular, this enables one to realise a combined regime, in which observing an interference pattern at a specified frequency within the resonance ensures high sensitivity and recording the resonant contour against frequency does wide dynamic range of measurements.
Besides, the elements of the apparatus may be arranged so that there are two interfering, with each other or with other beams as well, beams reflected from said metal layer so that the interaction of the medium under analysis with said sensitive material influences only one of the two said radiation beams, and each of participating in said interference radiation beams differs from other ones in position and/or direction in space. In particular, each of the two beams may be reflected from the metal layer under SPP excitation. In this case, the beams should be arranged so that only one of them undergoes reflection with SPP excitation at the interface of the metal and a sensitive material exposed to a medium under analysis. The use of two beams reflected from the same metal layer for their interference between each other or with a third beam enables one to reduce the influence of parasitic effects due to mechanical and/or temperature instabilities.
Besides, said facility for separating radiation is designed so as to yield said first and said second beam, with polarisation directions orthogonal to each other. In particular, there is a polariser (analyser) or a polarisation rotation means across, at least, one of said first and said second beam.
Besides, said source of electromagnetic radiation is designed so as to supply a divergent or convergent radiation beam on to said metal layer.